Conventionally, a recombinant protein is purified by allowing a target protein to be expressed as a fusion protein with an affinity tag and utilizing a specific affinity between the affinity tag and other molecule to thereby isolating the fusion protein from a soluble fraction. The affinity tag serves also to increase the solubility of the target protein to allow the fusion protein to be expressed as solubilized.
A commonly employed affinity tag may for example be His tag (histidine tag), GST tag (glutathione-S-transferase tag), MBP tag (maltose-binding protein tag), and FLAG tag. The His tag is a peptide containing about 6 to 10 histidine residues and specifically binds to a metal ion such as nickel ion. The GST tag and the MBP tag specifically bind to low molecular weight compounds such as glutathione and maltose, respectively.
For example, a solution of a protein tagged with the His tag is allowed to pass through a chelate resin having nickel ions immobilized thereon, onto which it is thus adsorbed. Subsequently, the nickel ion or a low molecular weight compound capable of binding to the nickel ion such as imidazole is allowed to pass through the resin, thereby allowing the fusion protein once adsorbed to be eluted and recovered.
The FLAG tag is a tag (epitope tag) utilizing an antigen-antibody reaction, and a protein tagged with the FLAG tag can be isolated from the soluble fraction by allowing it to bind to an anti-FLAG antibody. The epitope tag may otherwise be myc tag and HA tag, and the aforementioned His tag and GST tag can also be employed as epitope tags.
Such an affinity tag is designed also for allowing the fusion protein to be cleaved into a target protein and the tag upon a protease treatment thereby separating the tag from the target protein. For example, a fusion protein once adsorbed onto a resin via a tag is subjected to a protease treatment to separate the target protein and the tag from each other thereby allowing the target protein to be released and recovered exclusively from the resin.
Patent Document 1 discloses a technology for reacting a protein, which was labeled with a composite tag consisting of a His tag and a FLAG tag, specifically with a nickel-binding carrier and an anti-FLAG antibody-binding carrier thereby accomplishing separation and purification of the protein. Patent Document 2 also describes a method for recovering a target protein by using a peptide chain, which contains a cellulose-binding region of a cellulase, as an affinity tag and conduct a protease treatment to separate the tag from the fusion protein.